A continuous culture of human hepatoma cells has been used to study thyroxine-binding globulin (TBG) biosynthesis. The intracellular and secreted TBG are similar in size (MW about 54,000), indicating that there is not a large precursor form of TBG. A similar conclusion was reached from in vitro translation of mRNA from these cells; in this case, the nonglycosylated TBG had a MW about 44,000. A novel, apparently TBG-related, nonglycosylated protein (MW about 27,000) was also secreted by the hepatoma cells.